1. Goal: Biotech Assembler

The goal of Molecubotics is to develop the first assembler, using techniques
taken mainly from biotechnology.

Definition and importance of an "assembler":

The key bottleneck on the path towards realizing the full economic promise
of nanotechnology is to build an "assembler": a molecular robot that can be
programmed to construct molecular machinery made from the same kinds of
parts as it is (which can therefore construct copies of itself).

Having a working assembler system, even if it is very limited in the kinds
of building blocks it can use, permits the cost of molecular machinery to
drop to the cost of raw materials plus design, and permits rapid development
of improved assemblers and of many other new products.

Therefore, to develop the first assembler, choosing the fastest and simplest
strategy is much more important than choosing the kind of "building blocks"
used in the most desirable eventual applications. Once assemblers are in
hand, they can quickly be made to use better building blocks if necessary.

A biotech-based assembler of the simplest kind is envisioned to consist of
roughly 100 to 200 MBBs (Molecular Building Blocks), using an "alphabet" of
10 to 15 different types of MBBs. Some MBB types would be incorporated in
many places in the assembler structure. Having more MBB shapes available can
permit assembler designs containing fewer MBBs, especially after MBBs usable
as "rigid rods" are available.

Such an assembler would also need several (2 to 5) different "control
channels", i.e. parts whose state or position can be independently modulated
by outside signals sensed in parallel by all the assemblers dissolved in one
solution or attached to one surface. More complex molecular machinery,
developed later, could include control logic permitting a single outside
signal to control a population of assemblers, or permitting internal control
by on-board programs.

2. Requirements

Development of the first biotech assembler requires solving several
problems. We describe the problems here, and our strategies for solving them
in section 3.

2.1 Assembly (before an assembler is available)

Ability to assemble MBBs (Molecular Building Blocks) into precise, complex,
designed arrangements with up to hundreds of distinct component positions,
with a quick turnaround time for building newly designed structures from the
same kinds of MBBs.

2.2 Components

Several kinds of MBBs (synthetic constructs of modified proteins, DNA,
and/or small organic molecules), able to be assembled as in 2.1, and able to
serve the following functions:

structural framework

structural elements to hold small molecules together in the right relative
positions

flexible parts (tethers, non-rigid point-to-point connections)

actuators and/or motors

parts with application-specific functions, including optical, electronic,
catalytic, ligand recognition (for an assembler, this includes tools for
grasping and joining the MBBs being assembled)

at least some parts whose structure or activity is sensitive to external
"control signals", which can be supplied in parallel to many molecular
machines, and which can be rapidly varied in a controlled sequence.

The functions above are sufficient to construct an assembler; many potential
earlier products would require only some of these functions. For every
required MBB function, there are known biological or organic molecules able
to serve it, when slightly modified using known techniques.

Some of the biomolecules able to serve in MBBs with various functionalities
are: <table not yet here, but we have it elsewhere ###>

The application-specific MBB functionalities required by an assembler
(grasping and MBB-attachment tools) are given separate requirements sections
below.

Farther ahead, components which support additional functions would be
useful:

support for "microenvironments" (e.g. cages, membranes, seals, pistons, gates, pipes),
which is desirable for advanced applications, such as assemblers which can
handle non-biotech materials

components for internal computation and logic, and for sensing the
internal state of the assembler

2.3 Grasping Tool

Any assembler needs a way to hold onto its "workpiece" (the MBBs it has
already joined together to form part of its product), and to actively move
new MBBs into the desired position and orientation relative to the
workpiece, before attaching them to it.

Holding the workpiece is unnecessary if both it and the assembler are bonded
rigidly to some substrate. It is more likely that the first assembler will
either be fully dissolved, or will be insufficiently rigid for this to work.
In that case, the assembler will need to move the new MBB relative to the
workpiece while holding both of them.

Depending on the relative strength of the grasping bond, bonds within the
workpiece, and the assembler's actuators, the tools for grasping either the
new MBBs or the workpiece may need to have a grasping affinity which can be
modulated in some manner.

2.4 Attachment Tool

After an assembler moves a new MBB to its desired place in the workpiece, it must
attach it to the workpiece before it can place more MBBs. This requires that the
MBB stick more strongly to the workpiece than to the grasping tool ("hand")
which is holding it, so that it stays in place when the hand is moved away.

If the new MBB-workpiece bond is not strong or robust enough for the final
product, it must also later be strengthened or augmented by a better bond,
but this might be done as a separate processing step for the product as a
whole, rather than by the assembler per se. That is, there is a basic choice
of whether MBB attachment is a 1-step process (where the assembler makes the
permanent MBB-workpiece bond before releasing each MBB) or a 2-step process
(where the assembler only makes a temporary bond, good enough to allow it to add more
MBBs to the workpiece).

The choices for MBB attachment bonds and MBB grasping tools (hands) interact
strongly due to the need for the hand to leave the MBB on the workpiece when
it moves away. This is the issue most likely to favor making an immediate
permanent bond between MBB and workpiece, since then (as described in
section 3.5) the hand can probably just pull away without down-modulating
its affinity for the MBB. However, making permanent bonds quickly is a
nontrivial problem, as discussed in section 3.6 (Attachment Tool Milestone).
So the first assembler might make use of either of two basic schemes:

attach MBBs to the workpiece weakly, down-modulate the hand affinity
before pulling it away after adding each MBB, and after the workpiece is
fully assembled, strengthen all MBB-MBB bonds at once, using a high-yield
but perhaps slow reaction;

or, after attaching each MBB to the workpiece, bond it into place using a
high-yield, fast reaction, and only then pull the hand away, with no need to
modulate its affinity for the MBB.

(Note that the first scheme is similar to the "DGAP" scheme (section 3.1.1)
for using DNA-guided self-assembly to arrange MBBs before an assembler
is available, followed by a non-specific slow reaction to strengthen all
MBB-MBB bonds at once.)

See section 3.6 (Attachment Tool Milestone) for discussion of how to meet
this requirement.

2.5 Macroscopic Interface

A useful assembler system consists of a large number of discrete molecular
machines (assemblers), constructed as in 2.1 from parts in 2.2, mounted or
enclosed in some manner which permits:

control of their overall environment

provision of operational power or fuel

provision of MBBs to be incorporated into whatever the assemblers build
(more assemblers or other products)

provision of complex sequences of control signals which determine what
each assembler constructs, as they operate in parallel

extraction of products

some observation or sampling of the assemblers' structure and activity as
they operate, for debugging

The main choices for the operating environment of a population of the first
kind of assemblers will be:

dissolved in solution, in a manner permitting rapid replacement of the
solution, e.g. by filtering;

dissolved, but also tethered to a solid support, such as a powder or beads
in a column, or the polymer chains in a gel, or a solid surface;

in solution, but attached to a flat solid surface.

The latter choice should permit viewing of the assemblers and their
products by an AFM operating in solution, and it might even permit
influences by the scanned probe on individual assemblers (mainly useful for
debugging).

2.6 Design of Assembler Systems

The first assemblers will probably be dissolved in a fluid, or attached at
random positions to a common substrate, but later ones may benefit from
being attached to each other and organized at larger spatial scales, and
from containing different specialized machines working together, some of
which perform internal computations which control the others.

This is why we speak not just of "assemblers" but of an "assembler system",
which is like an "operating system for molecular machinery" (analogous to a
software operating system), and also somewhat like an "artificial cell or
organism".

The design of an assembler system will ultimately be much more complex than
that of each individual machine it contains. But the simplest systems
(identical assemblers controlled externally and in parallel) will work well
enough to get through the "assembler bottleneck" (section 1) and greatly
speed up further development.

One requirement is to design the first assembler system, including all the
assembly instructions needed to make the first assemblers "from scratch",
and to design the control signal sequences which cause those to make more
assemblers, other products, or more advanced assemblers.

Since even the first assembler may contain a few hundred MBBs per copy, its
design is likely to benefit from both off-the-shelf and custom-built
software tools, for CAD and for analysis and simulation at various levels,
covering not only atomic structure and chemical properties of components in
detail, but also mechanical and thermodynamic properties of components
treated as units. Development of this software is also a significant
requirement, especially since improvements beyond the very first assembler
are more likely to be limited by the speed of generating new designs to try
than by the speed of instructing the existing assemblers to build them.

3.1: Assembly Milestone: controlled assembly of small aggregates of MBBs

As explained in our document
Biotech as the fastest pathway to an assembler,
finding methods to assemble simple aggregates from well-defined MBBs with
high spatial and geometric control is the most fundamental and important
problem that is holding back substantial progress in nanotechnology. Thus,
this milestone is the most urgent. It will be achieved when:

(1) At least one kind of MBB has been developed, which can be varied in
small ways (i.e. very similar MBB types can be developed) in a rapid and
reliable manner; the MBB might consist of a simple synthetic construct of a
specific protein, some DNA, and perhaps small organic groups, or it might be
a pure-DNA construct such as a "double crossover molecule".

(2) MBBs of this kind can be reliably assembled into small, well-defined
aggregates, containing 5 to 10 MBBs each, in a precisely controlled
arrangement, which can be altered by design in a straightforward and general
manner.

Since most development work awaits this basic ability to assemble small
structures, several methods should be tried in parallel. Some of the methods
we think can be most quickly developed include:

3.1.1: DGAP, or DNA-Guided Assembly of Proteins

This requires each structural MBB to have different DNA sequences
at several specific locations on its surface, as well as chemical
groups which can be covalently bonded to lock the arrangement of MBBs
into place once it has self-assembled by DNA hybridization. The same
MBBs, but with different DNA sequences, would form different structures.

(DGAP is the subject of a 4-page "description of invention" notarized in
January 1994, and is described in detail in our 1996 document A Proposed Path from Current Biotechnology to a Replicating Assembler;
several possible ways to make MBBs suitable for DGAP are described in our
1997 document Production Strategies For Molecular Building Blocks
Suitable For DGAP. One of the simplest ways makes use of biotinylated
DNA attached to the protein Streptavidin; we have computed specific DNA
sequences which should permit this method to work. Unlike other proposals
for joining MBBs using Streptavidin and biotin, or indeed unlike most
proposals for using DNA to build nanostructures, we propose a method for
selecting only one of the several possible arrangements of 4 different DNA
sequences in the 4 locally identical binding sites on Streptavidin; this is
crucial for obtaining specific structures when these MBBs are used in
complex aggregates.)

3.1.2: Use of arrays of DNA double-crossover (DX) molecules

Seeman and
Winfree have shown that periodic 2-dimensional arrays of DX molecules can be
formed by DNA hybridization. In principle, it should be straightforward to
extend this procedure to more complex patterns, to bounded rather than
periodic patterns, to 3 dimensional arrays, and to use chemically modified
DNA with more desirable properties.

(For double crossover (DX) molecules, this milestone is close to having been
already achieved by Seeman & Winfree; however, for many uses it will be
necessary to modify DX-based MBBs to give them improved strength, altered
chemical structure, and/or attachment points for other kinds of MBBs based
on proteins. For this reason, even after achieving this milestone with DX
molecules, it may be worth pursuing it with other kinds of MBBs.)

3.1.3: Misc.

There are a variety of other possibilies to explore, which we have
not yet studied in as much detail. For example, some kinds of proteins can
form 2D or 3D crystals, accessible by scanning probe (AFM), to which other MBBs
could be weakly attached, and then removed from selected positions by some
modification performed by a scanning probe.

3.2 Scaffolding Milestone: assembly of hundreds of MBBs

Once small aggregates of MBBs can be routinely put together, this should be
scaled up to larger structures consisting of up to hundreds of MBBs.
Depending on how well the small aggregate technology works, this may require
optimization of MBB purity, of MBB-bonding yields, of assembly sequence or
speed, and/or of stability of MBB bonds (e.g. by adding additional
crosslinks).

It may also be desirable to develop a wider range of MBB shapes (especially,
long rigid rods) to reduce the parts count of structural MBBs required for
the scaffolding or framework of each assembled structure.

This milestone will have been achieved once large aggregates can be
routinely synthesized and characterized.

This milestone can be worked on in parallel with the development of more
kinds of MBBs and more uses for small aggregates. If the large aggregate
milestone takes too long to achieve, there are ways to develop an assembler
which don't require them. However, the selectivity of DNA hybridization is
in theory sufficient to permit thousands of parts to be joined in a unique
arrangement.

3.3 Actuator Milestone

Any interesting machine needs movable parts and ways of moving them, i.e.
actuators and/or motors. Developing and testing at least one actuator
usable in an assembler is another important milestone.

There are several actuation methods possible using DNA, some of which have
been published by others and one of which is (to our knowledge) proprietary
to us. There are several protein-based molecular motors that the research
community is studying in ever more detail, all of which could potentially be
used in machine designs. There are a variety of proteins and protein domains
whose shape is changed by binding of small ligands. Other methods are mentioned
below.

A requirement for an actuator to be useful in a primitive assembler, often
not appreciated, is that multiple actuators in one device can be controlled
with independent external signals. For example, if proteins which change
shape in response to small ligands are used, several *different* proteins
(which respond to different ligands) will be needed in each machine, to
provide it with enough independently controllable degrees of freedom. This
suggests that actuator designs which use specific DNA sequences for control
will be the simplest to develop for this purpose, since only a single design
(with a DNA sequence which can be easily varied) must be developed.

Achievement of the assembly milestones (3.1 and 3.2) will help greatly with
building and testing actuators, since they need to be anchored in a
structure, much like muscles must be anchored to a skeleton. Some results
might be achieved earlier, however, with actuators suspended between
scanning probe tips and substrates, or between glass beads suspended by
optical tweezers, both of which methods have been used (for example) to
measure forces related to DNA hybridization or stretching.

This milestone will have been achieved when at least one actuator sufficient
for use to control an assembler (which permits multiple actuators in one
machine to be independently controlled) has been constructed and
experimentally characterized, and when it is made compatible with the MBB
assembly method developed in 3.1 and 3.2. Parameters such as force
generation and positioning accuracy need to have been measured at least in a
crude manner, along with error estimates for those values. It will also be
important to know with what cycle time the actuator can be moved to a
different position, and how this might be optimized further if necessary.

More advanced (internally complex) assemblers can take advantage of faster
actuators even if fewer independent control signals are possible for them,
so control methods other than DNA (which diffuses relatively slowly due to
its size) should be explored even if DNA control signals are sufficient for
the first assembler. Even the first assembler may benefit from a few fast
control channels in addition to a larger number of slower (DNA-based)
control channels.

Besides protein shape modulation by small ligands, fast actuation of many
machines in parallel might be possible by variations over time of
temperature, ionic concentration (including pH), electric field, tension or
compression in a substrate, or other variables. (One exception is solution
pressure, which doesn't appear easy for biomolecule-based actuators to
respond to, since the biomolecules I'm aware of show too little volume
dependence on pressure. Eventually it might be possible to use proteins
embedded in the surface of a gas-containing vesicle as a pressure-sensitive
actuator.)

There are also molecular motors sensitive to variation of ionic
concentration across a membrane, and membrane-embedded ion-channel proteins
sensitive to the electric field across a membrane. One such ion channel has
been engineered (by Micah Siegel) to contain green
fluorescent protein whose fluorescence differs when the channel is open or
closed; reengineering such proteins in other ways might be used to develop
other kinds of actuators or transducers.

There are also possibilities for actuation of individual machines (one at a
time) using electric current in a wire built into a substrate, optical
tweezers moving micron-sized beads covalently attached by tethers,
influences by scanning probe tips, and other methods.

3.4 Simple Machine Milestone

Once an actuator is developed, several can be assembled into one device,
which will constitute a simple machine, with a few degrees of freedom of
control.

Such a device would probably require 10 to 50 MBBs, and would therefore
require prior achievement of the Scaffolding milestone (3.2). This is also
the level of complexity at which custom software tools are likely to become
especially important for design and simulation.

This milestone will have been achieved when a simple machine can be
constructed, controlled, and proven to move as expected when under control.

(Relative positions of parts in each machine can be monitored statistically
(averaged over all machines in a large sample being controlled in parallel)
by the change in fluorescense of optically active molecules depending on
their separation, and perhaps by other methods such as NMR. Direct
observation of individual machines by AFM might also be possible, depending
on the machine design.)

3.5 Grasping Tool Milestone

As described in 2.3, assemblers must grasp both their workpiece and the new
MBB they want to attach to it. There are several conceptually different ways
this might be organized. The workpiece and the new MBB might be grasped by
different methods, using specialized sites on the workpiece, or by the same
methods, so the assembler needs only one kind of grasping tool, or "hand"
(though it would need two or more instances of that hand in each assembler).
New MBBs must be grasped and released many times during a product's
assembly, whereas the workpiece might be grasped and released only once --
or the assembler might continually move along the workpiece (especially for
workpieces larger than the assembler), or regrasp the workpiece at a new
site before adding a few MBBs near that site.

We will focus here on the simplest schemes which might be adequate for the
first assembler to make copies of itself, even though the more complex
schemes will be very useful for extending what the assembler can build.
Accordingly, we'll assume that the workpiece can be grasped on the same
kinds of sites and by the same kinds of "hands" as the new MBBs can, so we
can limit the discussion to the hands for grasping new MBBs.

One conceptually simple grasping scheme is to have a single hand for
grasping new MBBs, whose affinity for a specific part of the MBB can be
controlled by an external signal (such as the concentration of a ligand
which binds somewhere else on the hand). MBBs added to solution diffuse onto
this "hand" and stick there in a controlled orientation (see section 3.6 for how they can
avoid prematurely sticking directly to the workpiece); the hand is moved
to the desired position using other control signals; the MBB is then joined
to one or more other MBBs already in the workpiece (section 3.6); then the
hand control signal is modulated to make the hand release the MBB. (Which
kind of MBB the hand grabs is determined by adding only one type at a time
to the solution.)

The affinity of the hand for the MBB it grasps might be modulated by the
concentration of a small ligand which binds to a protein in the hand, by DNA
which binds to complementary DNA in the hand, by moving a "fake MBB" built
into the assembler into a position where it can compete with the "real MBB"
for binding to the hand, or by other means.

A simpler scheme, which avoids any need for modulating the hand-MBB
affinity, is for the hand to just pull away from the MBB after it is joined
to the workpiece; this requires that the hand-MBB affinity is weaker than
both the MBB-workpiece bond and the actuators used to move the hand. We
expect that this scheme will be adequate for the first assembler, but even
so we will need to compare the extra complexity of affinity-modulated hands
with the easier requirements on MBB attachment methods which their use would
permit.

Due to the relationship among the choices for grasping method, MBB
attachment method (3.6), and the actuators for moving the hand, these
components can't be designed in isolation. It is also possible that the
first assembler will need two different kinds of hand so it can handle two
classes of MBBs which are grasped in different ways. Even so, it is useful
to make a separate milestone for developing a grasping tool.

The grasping tool milestone will have been achieved when:

for each kind of MBB needed as a component of the first assembler, it is
known by what kind of hand it will be grasped;

it is known what kind of hand will grasp the workpiece, and the
substrate it will be built onto (if there is one);

for each necessary kind of hand, the mechanical and thermodynamic
characteristics of the hand/MBB grasping bond (affinity, force-displacement
relationship, equilibrium relative position and orientation) are measured
well enough for modelling the assembler and designing its control sequences
for grasping and moving MBBs using that hand;

for each kind of hand, and given the choice and study of the MBB
attachment method (3.6) for the MBBs it grasps, the process of pulling the
hand away from the MBB (after down-modulating the hand's affinity for the
MBB, if necessary) can be predicted to break only the hand-MBB bond (and not
the hand's actuator, the hand, the MBB, or the MBB-workpiece bond), with
sufficient reliability that the average number of errors during the assembly
of each complete product is enough less than 1 that the yield of completed
products is high enough.

It is worth noting that since the first assembler will necessarily have a
structure which can be put together just by self-assembly, it will have an
easier job than described here when making a second assembler with the same
structure, whose MBBs could after all just self-assemble into the correct
places. That is, there is a tradeoff between how much the correct
positioning of each MBB in the product is controlled by that MBB (e.g. by
the DNA sequences attached to it), and how much needs to be controlled via
motion of the assembler's hands.

It may prove simplest to let the first assembler gain much assistance from
MBBs capable of self-assembly, since this would permit it to work even with
crude positional control of MBBs, and no orientational control; even then,
use of an assembler would permit more complex products than could be
produced with self-assembly alone. However, as soon as the assembler itself
does most of the work of arranging, orienting, and joining the MBBs in the
right way, the MBBs themselves can be much simpler, and the objects made
from them can be much larger and more complex.

3.6 Attachment Tool Milestone

(See requirement 2.4 for the problem to be solved and for two different
general strategies for solving it.)

There are many possible methods by which an assembler might attach a new MBB
to its workpiece; we don't yet know which one will be simplest to develop,
but any of them might turn out to be difficult. Of the problems to be
solved, this one has the most uncertainty about the nature of the best
solution. Accordingly, it is necessary to study several possible ways:

3.6.1 Covalent bond formed upon contact, perhaps only under certain solution
conditions

Problems: if the reaction is fast, MBBs might attach to undesired locations
on the workpiece before they became bound to the "hand", but if it's slow,
the assembler will need too much time to attach each MBB. There are many
reactions to consider, but many of them are likely to be slower than ideal.

Possible solutions (to slowness and/or undesired attachment points):

speeding the reactions using catalysts built onto movable parts of the
assembler (perhaps metal ions held in protein domains, perhaps evolved
enzymes) should be investigated extensively, as this method would produce
the best outcome if it worked well, and one which can be generalized to the
building of a wide variety of structures. Bonds in the wrong locations would
be prevented by not moving the catalysts into those locations; note that the
catalysts can still be allowed some positional freedom so it is easier to
move them close enough to the reaction site. Alternatively, catalysts can be
added to the solution only after the MBB is in the right location.

protective groups on either the workpiece or the new MBBs could be removed
by the assembler only after it has grasped the new MBB, to prevent
attachment at the wrong locations. Ways to remove protective groups include
changing solution properties, moving them to parts of the assembler with an
affinity for them (down-modulated later to get rid of them) or which
catalyze their destruction, or adding competitive substrates for them to the
solution.

another attachment method, faster but weaker, can be used temporarily,
allowing a slow reaction to form permanent bonds without delaying the
assembler's placement of the remaining MBBs.

reactions which require an extra reactant from the solution can be
delayed, by not adding that reactant to the
solution until the MBB is in place.

3.6.2 DNA Ligase

If DNA is used as a temporary attachment method, it might be possible to
use DNA ligase (perhaps tethered to a movable part of the assembler) to make
such bonds permanent; note that the effective concentration of an enzyme can
be made quite high by holding it in the right place without much positional
freedom, which can speed up the reaction rate.

3.6.3 DNA Cross-linking

There are chemical groups which can be built into DNA which form
permanent bonds when held near each other, as they would be by DNA
hybridization; some of these require photoactivation, and others require
only time and appropriate conditions (e.g. "Glick bases" which form
disulfide bonds). Such groups could be built into an ssDNA strand near the
point at which it is attached to an MBB, providing a short connection length
between two such MBBs.

3.6.4

Note that several tether-like connections between MBBs can result in a
rigid connection if they are properly arranged around a surface on which the
MBBs make intimate contact. Also, in many products, many of the connections
between MBBs need not be rigid, when all that's required for product
operation is sufficient nearness between connected MBBs. This is often the
case, even for most components of an assembler (the main exception being an
outer rigid framework).

The most attractive connection method in the long run is likely to be the
use of catalysts, such as evolved proteins, moved near the reaction site by
the assembler, to speed up covalent bond formation requiring no extra
reactants. But it's not clear whether this can be developed sooner than one
of the less ideal methods.

This milestone will have been achieved when:

at least one scheme for solving simultaneously the requirements for
grasping, attachment, and actuators, has been developed, in which the
attachment reactions (both permanent, and temporary if there is one) have
been characterized for rate and yield when used under the same conditions
and similar reactants as in the assembler;

the reaction rates and yields are high enough to permit their use for
sequentially joining many MBBs into one product.

If yields are not high enough, products can be designed with redundant
connections between MBBs, and/or from subassemblies built in parallel, which can be
tested so that only the correct ones are further assembled into final
products.

3.7 Simulated Assembler Milestone

This section is not finished ###. Outline: it is desirable to simulate the
operation of assemblers like the one to be built first, and useful even at
prior stages, in order to understand the tradeoffs between various MBB
limitations, as well as to explain and demonstrate the basic idea of what we
are trying to achieve. Since any assembler is too large for atomic-level
simulation, it
must be simulated mainly at a higher level, more like the level on which its
designers or molecular biologists would think about it, corresponding to the
mechanical and thermodynamic properties of its components, treated as units.

3.8 The First Biotech Assembler

The real test of the biotech assembler-development methodology will be to
build the first machine that can construct copies of itself when
controlled by external signals.

This will require a moderately complex design, probably containing on the
order of at least 10 actuators and 200 MBBs (though these are very rough
estimates, and there are tradeoffs between MBB count, MBB variety, and
design software complexity).

The development of the first assembler will use the results of all previous
milestones, though most of those should be worked on
in parallel, so that design tradeoffs can be
made which permit the first assembler to be built sooner.

This milestone will have been achieved when the number of functioning
assemblers in our laboratory grows over time, since new ones are constructed
faster than old ones deteriorate, even when the yield of harvesting new
assemblers and placing them into operation in a new (or the same) operating
environment is taken into account.

Depending on the properties of the technologies developed to reach the prior milestones,
achievement of this milestone may also require optimization of actuator
speed, of MBB lifetime, or of the bonds used to attach new MBBs to the
assembler being built.

4. Potential Spin-off Products

We have not yet analyzed these suggested product opportunities in any
detail, and doing so would require significant effort, but we think that the
proposed basic technologies are general enough that many profitable
applications could be found, most of which we have never thought of.

One way to look for product opportunities would be to search the literature
in various nanoscale-related fields for attempts to make minor
nanometer-scale improvements to various materials or processes, where there
is an implication that this would have an economic payoff, and then to
determine the intended application, and think of ways to make better
improvements for the same application.

We organize these potential product ideas by the milestones in section 3
which would need to be achieved before the products could be developed. For
each milestone, we assume that all lower numbered milestones have also been
achieved. In all cases, further development would be needed which was
specific to these products, and which would often require different skills
and equipment than the assembler project milestones themselves.

Some of the products listed here would only be practical if large cost
reductions could be achieved in synthetic DNA and proteins for use in building blocks.

Note that applications of large crystals of small MBB aggregrates are listed
here, whereas applications of large complex MBB aggregates (whether formed
into crystals or not) are reserved for the next section.

- "Molecular tips" and receptors for SPMs: Constructs of several MBBs could
be designed for attachment to the end of a scanning probe microscope (SPM)
tip, to provide it with several different molecular probes (sub-tips, and/or
receptors) of known atomic structure and relative position. Such "molecular
tip" devices should greatly expand what can be measured and perhaps modified
by scanning probe tips, and especially the reliability of such operations,
and the tip lifetime. Thus they will be useful for research and perhaps
(using tip arrays) for manufacturing. They also have important internal uses
for helping Molecubotics do nanoscale research, and possibly for making
highly complex assemblies of MBBs "one at a time" (rather than in parallel
in a solution.)

- Protein purification: several receptors with affinity to different parts
of a protein surface, when arranged around one site with a controlled
spatial relationship, chosen by design, would have a much higher affinity
for that protein than any individual receptor had. This could be used for
exceptionally high-quality purification of that protein. We presume there
are markets for purification of proteins meant to be used as pharmaceuticals
or in their production, as well as proteins used in biotech research.

There is also an important internal use, since making complex machinery from
protein-based MBBs demands very high purity in the base proteins, to avoid
incorporating defective MBBs at any single point in the design. A related
internal use is to make protein-based MBBs at much lower cost than the
present cost of their base proteins, for base proteins which are available
in large quantities, but only in a form which is presently too expensive or
difficult to purify.

- Small molecule purification: the same idea can be applied to the affinity
purification of small molecules, by building "cages" of MBBs which surround
sites intended for those molecules with multiple groups with affinity for
them. This idea has already proved fruitful when the cages are
custom-synthesized large organic molecules; our methodology would provide a
simpler systematic way of developing new cages more quickly.

- Artificial zeolites for catalysis: zeolites are useful as specific
catalysts because they contain many "cages" of the same internal structure.
Another use for "MBB cages" (especially when designed to have specific
functional groups on the inside surface of the "cage") would be to ease the
design of cages for use as catalysts. Cages synthesized by more traditional
methods are already an active area of research in the chemical industry,
achieving some success.

- Protein crystallization aids: "cages" made of several MBBs, surrounding a
protein of unknown structure (but attached into the cage as if it was also
an MBB), could be designed to crystallize, independently of whether the
uncaged central protein would crystallize on its own. The resulting crystal
structure, if it can be determined to atomic precision by X-ray diffraction,
would provide an atomic structure map of the central protein as well. This
has markets in pharmaceutical research, since many proteins of interest
can't be crystallized directly. Seeman has long had the same application in
mind for extended scaffoldings made of DNA. DX molecules extended to work in
3D and to have holes (which Seeman has already proposed) are especially
attractive. MBBs other than pure DNA may (or may not) prove easier to use
for this.

- MBB kits: the MBBs able to be assembled by our technology could be
directly sold, for research or for use in products developed by other
companies.

- Biosensors: some existing products consist of complexes of a small number
of proteins, such as an antibody and several enzymes, which are able to
generate a visible signal (e.g. if the enzymes generate a product which
appears blue) at the locations to which the antibodies attach; these are
used as probes for the locations of antigens in tissue samples or gels. It
seems likely that greater spatial control or higher parts count provided by
our methods could be used to optimize some such applications or to develop
new ones.

- Protein-based pharmaceuticals might be more reliable if the proteins were
arranged in a precisely designed manner, with controlled relative position
and orientation.

- Macroscopic filters: Large crystals of small aggregates of MBBs might be
used as filters whose pores would be relatively large and have a precise
internal structure, able to be functionalized with chosen molecules in a
designed way. (For this to be a practical method for large-scale
purification, it would probably be necessary to improve MBB lifetime and
lower MBB cost, in ways described above.)

- Macroscopic materials with optical, electronic, or electrochemical
activity: Large crystals of small aggregates of MBBs, carrying functional
groups with optical, electronic, or electrochemical activity, might be able
to be developed into improved materials for use in batteries, fuel cells,
photovoltaic cells, or other optical, electronic, or electrochemical
devices, where the ability to achieve a precise arrangement of the active
functional groups is presently a limiting factor, and when the device
operation is compatible with the type of MBBs used. Making this practical
might require development of non-protein MBBs, and will probably
require ways to improve MBB lifetime. It might also require surface
modifications to remove undesired activities, or complete removal of water
from the final product, if either of these interferes with the intended
activity of the device (which seems likely). It would also be necessary to
reduce MBB cost in ways described earlier.

- Scaffolding for molecular electronics: several single-molecule
electronic devices have been proposed. Seeman and Robinson (1987) have
suggested using DNA as a scaffolding for organizing such molecules. Another
way would be to build complex aggregates of many MBBs, able to form a 2D or
3D crystal, with the MBBs having surface receptors for specific molecular
electronic devices, and also for the ends of molecular wires which would be
used to connect them. If this was the first practical method for making
complex molecular electronic circuitry, or if it had some significant
advantage over other methods, the potential market for assembling high
capacity computers and memories would be very large.

Protein lifetime might be an issue in this application (assuming the
device-receptors are protein-based), so the lifetime might have to be
extended by removing water, crosslinking the proteins, adding polymers to
form a non-protein permanent matrix, or by some other means.

5. Project Organization

<section not finished ###>

(proceed with most milestones in parallel)

(note that the best assembler designers are likely to be designers of computer
software, or hardware, or mechanical objects, who know some biotech and can
talk with its practioners; the Foresight community is a good source of such people.
Of course, to develop the components and basic technologies requires people
who are already skilled in the application of biotech methods.)